Improved understanding of cellular function as well as disease pathology provides many possible targets for developing new therapeutic and diagnostic reagents. A general problem with turning this cellular knowledge into treatments is that many potential targets are functionally undruggable. Hopkins et al. (2002) Nat. Rev. Drug Discov. 1:727. Therefore, it is not possible to develop small molecules that can modulate many of the protein-protein interactions that control cellular function.
Monoclonal antibodies provide an important route to target protein surfaces and represent both a large and growing portion of approved therapeutics. Nelson et al. (2009) Nat. Biotech. 27:331. However, antibodies are expensive to produce and dose, difficult to administer and control quality, can produce neutralizing immune responses, and cannot be used to target intracellular proteins. Cho et al. (1996) Trends in Biotech. 14:153. The fact that such cumbersome molecules dominate therapeutic protein-binding reagents demonstrates both the importance of these reagents and our desperation at finding viable replacements.
Peptides provide an attractive alternative route to developing this type of ligand. Non-ribosomal proteins such as cyclosporine are peptidic compounds that incorporate amino acids beyond the 20 common residues coded for by DNA. These molecules are capable of modulating intracellular protein-protein interactions (PPI), and exhibit oral bioavailability. However, there have been a limited number of therapeutic molecules derived by searching natural products. Ho, S. et al. (1996) Clin. Immunol. And Immunopathol. 80:S40; Baumann et al. (1994) Protein Sci. 3:750; and De La Cruz et al. (1996) Biochem. 35:14054.
Selection methods such as mRNA display and phage display have been shown to be capable to generating peptidic ligands able to bind to and modulate PPI for a very large variety of protein interactions. Ja et al. (2004) Biochemistry 43:9265; Ja et al. (2006) ACS Chem. Biol. 1:570; and Millward et al. (2007) ACS Chem. Biol. 2:625. However, these peptides suffer from poor bioavailability due in large part to proteolysis.
A more traditional approach to peptide medication and drug development requires the generation of a lead molecule followed by extensive medicinal experimentation. Present current strategies include helix stapling, peptoid synthesis, beta-peptide generation, and N-methyl incorporation. Fiacco, S. V. and Roberts, R. W. (2008) ChemBio Chem 9:2200; Walensky et al. (2004) Science 305:1466; Miller et al. (1994) Bioorganic & Medicinal Chem. Letters 4:2657; and Nguyen, J. T. et al. (1998) Science 282:2088. However these techniques are very labor intensive, often taking years, with no guarantee of success. Many times the resulting product will exhibit a loss in function, specificity, or both.
Previous attempts to stabilize and enhance protease stability of a peptide include chemically scanning each of the 9 positions with an N-methyl analog. Four (4) of the 9 positions enhanced protease resistance indicating that a single N-methyl provided a window of protection around the scissile bond. The resulting protease resistance was dramatic, ranging from 70 fold to over 1000 fold at the site of cleavage. Unfortunately only one of the 9 substitutions retained the same binding specificity as the parent molecule; 4 of the sequences lost function while 4 had altered specificity now binding the related protein Gα12 instead of the original target, gαi1. Interestingly combining the two best Gα12 binding modifications resulted in a total loss of function (Fiacco et al. (2008), supra).
Another approach involved making covalent cyclic mRNA display libraries that had the unnatural amino acid N-methyl phenylalanine (NMF). That work showed that cyclic peptides could have antibody like affinity and that cyclization improved stability. However, the increase in protease resistance was modest (2.6 fold) and the resulting molecules lacked the unnatural amino acid (Millward et al. (2007) supra; Frankel et al. (2003) Chemistry & Biology 10:1043 and Gilmore et al., In Implementation and Redesign of Catalytic Function in Biopolymers; Springer Berlin/Heidelberg (1999) 202:77). Additionally Phe is a large residue and may be compatible in only a few of the positions.
Thus, a need exists to devise a scheme that retains the binding function of a natural peptide while dramatically improving its stability both in vitro and in vivo, that can be applied to any target of interest, thereby overcoming the limitation of the helix stapling methods described by Walensky et al. (2004), supra., which can only be applied to some helical structures.
A need also exists for new approaches to target unmet medical needs. Diabetes is an example. Diabetes can be divided into two categories. Type 1 diabetes is characterized by individuals with the inability to create insulin. This form of diabetes is a progressive disease characterized by significant loss in pancreatic β-cell mass leading to impaired insulin secretion. Impaired insulin secretion results in hyperglycemia which can lead to serious health problems such as ketoacidolysis if left untreated. Iyer, H., et al. (2010) Diabetes, Obesity and Metabolism 12:179. Type 2 is characterized by the inability to properly utilize insulin. Long term misregulation of blood sugar leads to an increased risk of heart failure, kidney disease, strokes, and limb amputation. Ford, E. S. Journal of Diabetes Ford, E. S. (Jul. 8, 2011). In the United States, more than 90% of individuals with diabetes have type 2 diabetes. WebMd 2011, available at the web address: diabetes.webmd.com/guide/type. Currently, diabetes is the seventh leading cause of death in the United States. Center for Disease Control 2011, available at the web address: www.cdc.gov/diabetes/pubs/estimates11.html.
According to the World Health Organization as of 2011, approximately 220 million people, or 3.2% of the world population, have diabetes. This number is expected to increase to 4.4% of the overall world population by 2030. Goldberg, M., et al. (2003) Nat Rev Drug Discov. 2:289. Diabetes becomes more prevalent with age, effecting 18.3% (8.6 million) of Americans over 60 years old (Center for Disease Control 2011, available at the web address: www.cdc.gov/diabetes/pubs/estimates 11.html).
There is a significant precedent for the development of peptides that are capable of binding protein surfaces with high affinity and specificity for the purpose of controlling protein-protein interactions in vitro. Ja, W. et al. (2006) ACS Chem. Biol. 1:570; Karatan, E. et al. (2004) Chemistry & Biology 11:835; Fuh, G. et al. (2000) J. Biol. Chem. 275:21486; and Stoop, A. A. and Craik, C. S. (2003) Nat Biotech 21:1063. However, their development into orally bioavailable therapeutics or therapeutics able to reach intracellular targets remains a tremendous challenge. This is due in part to the intrinsic difficulties these types of ligands face in crossing lipid bilayers. Morris, M. C. et al. (23001) Nat Biotech 19:1173; Goldberg, M. and Gomez-Orellana, I. (2003) Nat Rev Drug Discov. 2:289. There are examples pharmacologically relevant peptidic ligands that are thought to passively diffuse across cell membranes such as cyclosporine, amanitin, and phalloidin. Ho, S. et al. (1996) Clinical Immunology and Immunopathology 80:S40; Baumann, K. et al. (1994) Protein Sci. 3:750.; and De La Cruz, E. M. and Pollard, T. D. (1996) Biochemistry 35:14054.
Current biological treatments for diabetics typically center around the injection of various insulin products. Sales of insulin and insulin analogs resulted in over $15 billion in revenue in 2010. Currently, there is no FDA approved oral formulation for insulin. Developing an oral route to insulin administration would be a tremendous benefit to those affected by the diabetes, not only because of convenience, but also due to potential health benefits of this route of administration. After secretion from the pancreas, insulin travels to the liver before dispersion to the rest of the body. Orally absorbed drugs follow this same route; however drugs administered by injection do not. It is postulated that an oral treatment would alleviate adverse side effects of insulin administration such as weight gain and hypoglycemia. Funnell, M. M. (2006) Clinical Diabetes 24:154.
Effective treatment of diabetes also is hindered by low patient compliance. Today, insulin is administered by self-injection. An orally bioavailable drug would enhance patient compliance. However, orally administered drugs, such as peptidic ligands, are hindered by the facts that peptidic ligands must reach their targets to be effective diagnostically or therapeutically. Additionally, intracellular proteins must cross the plasma membrane and bioavailable peptides must traverse the intestinal mucosa to reach their targets. This invention addresses the limitations of the current state of the art.